Method of treating HIV in humans by administration of ddI and hydroxycarbamide

ABSTRACT

A method and composition for inhibiting the spread of a retrovirus such as HIV in a human cell population in which a retrovirus such as HIV is present has been found. The spread of the retrovirus is inhibited by treatment of the cells with a synergistic combination mixture of a dideoxy-ribonucleoside excluding AZT and hydroxycarbamide.

This is a continuation of application Ser. No. 08/169,253, filed Dec.20, 1993, now U.S. Pat. No. 5,521,161.

FIELD OF THE INVENTION

The present invention relates to a combination of a reversetranscriptase inhibitor and hydroxycarbamide in a synergisticallyeffective amount wherein the combination is useful in inhibitingretroviral spread.

BACKGROUND OF THE INVENTION

The expression "Acquired Immuno-Deficiency Syndrome" (AIDS) was firstused in 1981 to describe a state of cellular immune deficiency inhomosexuals, characterized by the appearance of opportunistic infectionsand Kaposi's Sarcoma evolving very aggressively (CDC (Center for DiseaseControl), MMWR, 30: 305-308.DC, (1981)). In 1983 a retrovirus sincecalled HIV (Human Immunodeficiency Virus type 1) was isolated among AIDSpatients (Barre-Sinoussi F. et al Science, 220: 868-870 (1983)).

Over the past several years, researchers and clinicians have gainedconsiderable experience in studying and caring for individuals infectedwith HIV throughout the often prolonged course of HIV disease and AIDS.On the basis of this experience, it has become clear that the pathogenicmechanisms underlying HIV infection and disease are not unidimensional,but rather are extremely complex (Fauci AS., Science, 239, 617,(1988)).Any attempt to design a comprehensive therapeutic strategy for HIVdisease must take this fact into account. (Fauci, 1993, Science,262:1011-1018).

After entry of the HIV virus into cells and uncoating of the HIVparticle, reverse transcription of the viral RNA genome into DNAreplicas occurs. Among several forms of unintegrated viral DNA (nowcontaining the long repeats LTRs!, at both the 5' and 3' ends), only thetwo LTR linear forms can integrate into the host genome. Such a processappears strictly dependent upon cell activation/replication of Tlymphocytes, although "resting" T cells are clearly susceptible to HIVinfection. (Zack J. A. et al. Cell; 61, 213-222, (1990)). Furthermore,part of the reverse transcription process also can occur in unactivatedT cells, a process that results in the accumulation of incomplete DNAmolecules, which may persist for several hours and remain capable ofbeing integrated into the host genome if the cell undergoes sufficientactivation (Zack J. A. et al, Cell; 61, 213-222 (1990)). Therefore,infected "resting" CD4⁺ T lymphocytes can be considered a transientviral reservoir in infected individuals (Bokrinsky M. I. et al; Science,254, 423-427, (1991)). These observations are of particular importancein anatomic compartments such as the peripheral blood and lymphoidorgans, where the CD4⁺ T cell subset represents the predominant infectedcell type (Schmittman S M. et al, Science, 245,305-308, (1989)); (FoxCH. et al J. Infect Dis; 164, 1051-1057, (1991)).

The above discussion provides a sound scientific basis for blocking theinitial burst of virus replication and dissemination as well as thepersistent replication throughout the course of disease by treatingHIV-infected individuals with antiretroviral agents from the earliesttime that HIV infection is recognized through the entire course ofinfection. Unfortunately, currently available agents are only partiallyeffective in suppressing virus replication and spread, and this effectis transient (Hirsch MS, et al, New Engl. J. Med. 328 1686, (1993)).Clear cut, but limited, benefit is seen when3'-azido-2',3'-dideoxythymidine or azidothymidine (AZT) is given to apatient with advanced HIV disease, and the benefits of earlyintervention are usually only temporary and do not result in significantlong-term advantages with regard to the course of disease and death.(Fauci, 1993, Science, 262:1011-1018).

A number of 2'-3'-dideoxynucleosides have been found to be useful forthe treatment or prophylaxis of retroviral infections and especially HIVand AIDS. Examples of such materials include: 2',3'-dideoxy-cytosine(ddC); 2',3'-dideoxy-adenosine (ddA); 2',3'-dideoxy-guanosine (ddG); and2',3'-dideoxy-inosine (ddI) and 2',3' dideoxy-thymidine (ddT). SeeEuropean patent application 0206497 and published PCT application numberWO 87/01284.

Hydroxycarbamide (HC) was initially synthesized over 120 years ago andhas been found to demonstrate activity against a broad spectrum oftumors. (Donehower, Seminars in Onocology, Vol. 19, No. 3, Suppl. 9(June) 1992: pp 11-19). Additionally, hydroxycarbamide has been used asa viricide. In published PCT application number WO 93/09718,hydroxycarbamide is taught to be useful in a hydrogel polymer coating ofa blood bag in order to inhibit viral and HIV infectivity.

Gao et al (PNAS, U.S.A., Vol. 90, pp. 8925-8928, October 1993) disclosethat hydroxyurea (hydroxycarbamide) treatment of peripheral bloodlymphocytes (PBLs) decreases dNTP levels and the DNA synthesis rate tolevels comparable to quiescent PBLs. The article alleges a possible useof hydroxyurea in AIDS therapy.

However, there still remains a need for more effective treatments forthe suppression of retroviruses and, in particular, the preventionand/or inhibition of HIV and viral spread. By viral spread, it isintended to include the inhibition of viral replication, and also mayinclude the ability of inhibiting the virus to infect a further hostcells.

Objectives of the present invention in the search for new antiretroviralagents include:

1) the identification of compounds with less toxicity and antiviralactivity greater than AZT.

2) the development of drug combinations which provide an additive orsynergistic effect and decrease the probability of drug resistantisolates.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a combination of a reversetranscriptase inhibitor and hydroxycarbamide in a synergisticcombination wherein the synergistic combination is capable of preventingand/or inhibiting the spread of retroviruses including HIV. Morespecifically, the present invention relates to a method of preventingand/or inhibiting the spread of retroviruses, including HIV (HIV-1 andHIV-2), HTLV-1, HTLV-2, SIV or HSV, by exposing a cell population,including cells infected by a retrovirus such as, for example, HIV, to asynergistic combination of a reverse transcriptase inhibitor andhydroxycarbamide. Additionally, the present invention encompasses thetreatment of HIV-infected and AIDS patients with a synergisticcombination of a reverse transcriptase inhibitor and hydroxycarbamide inorder to prevent and/or inhibit the spread of HIV in these patients.

In a preferred embodiment of the present invention, the reversetranscriptase inhibitors include dideoxynucleosides, such as, forexample, ddI, ddA, ddG and ddT (DT4).

In particular and in the preferred combination of the present invention,it has been found that a synergistic combination of hydroxycarbamide(HC) and 2',3'-dideoxy-inosine (ddI) can be formed which is especiallyeffective in preventing and/or inhibiting HIV spread. The preferredembodiment of the invention encompasses a composition including apharmaceutical composition comprising a synergistic combination of ddIand HC. The pharmaceutical composition can optionally contain apharmaceutically acceptable carrier and/or excipient and/or vehicle. Thepreferred method of the instant invention comprises preventing and/orinhibiting retroviral or HIV spread by treating a cell population,including cells infected with HIV, with a synergistic combination of ddIand HC. Additionally, the preferred method comprises treating an HIVinfected or AIDS patient with a synergistic combination of ddI and HC soas to prevent and/or inhibit HIV spread in the patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a study of the anti-viral activity of a mixture of ddI and HCon non-activated CD4+ lymphocytes infected with the HIV virus.

FIG. 2 is a study of the antiviral activity of a mixture of AZT and HC.

FIG. 3 illustrates the inhibition of viral spread by the mixture of HCand ddI in pre-activated peripheral blood mononuclear cells (PBMC)culture infected with the HIV virus.

DETAILED DESCRIPTION OF THE INVENTION

The following examples of specific embodiments of the present inventionare offered for illustrative purposes only and are not limiting withrespect to the scope of the disclosure or claim coverage.

Testing of the mixture of dideoxyinosine (ddI) and hydroxycarbamide (HC)on the spread of the HIV virus was conducted under two types ofconditions:

a) CD4+ lymphocytes purified from PBMC, and infected with HIV viruswithout prior activation/proliferation of these cells byphytohemagglutinin (PHA) and interleukin-2 (IL-2).

b) PBMC preactivated by PHA and IL-2, then infected with the HIV virus.

EXAMPLE 1

The activity of the mixture of ddI and HC on non-activated CD4⁺lymphocytes, infected with HIV virus was studied.

Non-activated CD4⁺ cells were infected, then treated for 7 days by HC,ddI or the combination of the two, then activated by PHA and IL-2(PHA-IL-2).

Cellular viability between 90% and 100% was observed during the firstseven days after infection, both for the infected control and for theinfected cells treated with the two drugs separately or in combination.Comparable proliferative cellular response was observed in the presenceof PHA-IL-2 for the first 3 days (days 7-9) both in the six virusinfected groups and in the non-infected, non-treated donor CD-4⁺ cells.This proliferative response is associated with cytopathic effect in theinfected control group, and in the groups treated with HC alone at 0.05and 0.15 mM: these groups had greater than 50% loss of viabilitycompared to the uninfected control group; this effect is due to viralreplication and is accompanied by large-scale production of p24-HIV inthe culture supernatant seen at day 15 (86215 pg of p24/ml for theinfected control, 75470 and 82005 for 0.05 and 0.15 mM HC treatmentgroups, respectively) see FIG. 1.

The cytopathic effect was observed later for the cells treated with 5 μMddI and reached substantially the same level of p24 production as theinfected control 10 days later at day 25 (101080 pg p24/ml), see FIG. 1.

The mixture of HC at 0.05 mM with ddI at 5 μM does not substantiallychange the viral replication profile as compared to ddI alone (84883 pgp24/ml at day 25), see FIG. 1.

By contrast, a surprising synergistic effect is observed with thecombination 0.15 mM of HC and 5 μM of ddI, where no residual viralproduction is detectable (<1 pg p24ml) at day 7 and day 25 despitecellular proliferation which is identical to the non-treated,non-infected control (>90% cell viability measured by MTT test).

FIG. 1, in particular, shows a study to the activity of a mixture of ddIand HC on non-activated CD4⁺ lymphocytes infected with the HIV virus.The CD4⁺ lymphocytes were purified from PBMC with immunomagnetic beads(Dynabeads® M450). These cells were infected with the HIV-1 virus strainIIIB at a multiplicity of infection of 5 000 tissue culture infectiousdose (TCID) per 10⁶ cells (241 pg/ml p24 antigen equivalent of virus).After 2 hours of virus cell contact, the cells were washed twice andplaced in the culture medium RPMI 1640 (supplemented with 10% fetal calfserum (FCS), 2 mM glutamine, penicillin 100 IU/ml and Streptomycin 100μg/ml) at a density of 1.3×10⁶ cells/ml. ddI was immediately added at aconcentration of 5 μM and HC at concentrations of 0.05 mM and 0.15 mM.The drugs and culture medium were partially renewed (50%) on day 4,maintaining the same concentration of each. On day 7, in order to removethe drugs, the cells were washed twice and put back in culture in thepresence of PHA at a concentration of 1 μg/ml and recombinant IL-2 at aconcentration of 20 U/ml. This culture was maintained until day 25, withpartial renewal (50%) of the medium twice a week. The number of viablecells was quantified by a tetrazolium-based colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)method (Pauwels, R. et al J. Virol. Methods, 20, 309-321, (1988)), andactivity is expressed as a percentage of the signal in the drug-free andvirus-free control. Viral replication was quantified by measuring theHIV-1 p24 antigen by ELISA using the Dupont de Nemours kit.

EXAMPLE 2

In order to determine whether or not this synergistic effect of ddI in amixture with HC is specific to ddI, or whether a similar effect could beobserved with AZT, a parallel study was conducted combining HC withazidothymidine (AZT) with surprising results.

As can be seen in FIG. 2, AZT alone at 5 μm has only slight antiviralactivity, less than 1 log, (88.4%) inhibition (10030 pg p24/ml comparedto 86215 pg p24/ml for infected control), less than ddI at the same doseunder the same conditions: 99.1% inhibition (766 pg p24/ml compared to86215 pg p24/ml for infected control, see FIG. 1). The drugconcentration used here are easily attainable in plasma (plasmaconcentration achievable under treatment conditions: 4 μM for AZT and 10μM for ddI (Yarchoan et al, New Engl. J. Med. 321 726-738,(1989)).

Comparable proliferative cellular response was observed afterstimulation by PHA-IL-2 in all groups. This proliferative response isassociated with cytopathic effect in the infected control group, and inthe groups treated with HC alone at doses of 0.05 and 0.15 mM andcombined with AZT at 5 μM (these groups had greater than 50% loss ofviability compared to the uninfected control group). The combination ofHC at 0.05 mM and at 0.15 mM with AZT at 5 μM (10108 and 9166 pg p24/ml,respectively) does not modify the viral replication profile compared toAZT alone (FIG. 2).

The results show that the synergistic effect which eradicates HIVreplication in CD4⁺ cells non-activated by PHA-IL-2 (Example 1) is notfound from the mixture of HC with AZT.

In FIG. 2, there is shown a study of the activity of the combination ofAZT and HC on non-activated CD4⁺ lymphocytes infected with the HIVvirus. The CD4⁺ lymphocytes were purified from PBMC with immunomagneticbeads (Dynabeads® M450). These cells were infected with the HIV-1 virusstrain IIIB at a multiplicity of infection of 5 000 tissue cultureinfectious dose (TCID) per 10⁶ (241 pg/ml p24 antigen equivalent ofvirus). After 2 hours of virus-cell contact, the cells were washed twiceand placed in the culture medium RPMI 1640 (supplemented with 10% FCS, 2mM glutamine, penicillin 100 IU/ml and streptomycin 100 μg/ml) at adensity of 1.3×10⁶ cells/ml. AZT was immediately added at aconcentration of 5 μM and HC at a concentration of 0.05 mM and 0.15 mM.The drugs and culture medium were partially renewed (50%) on day 4,maintaining the same concentration of each. On day 7, in order to removethe drugs, the cells were washed twice and put back in culture in thepresence of PHA at a concentration of 1 μg/ml and recombinant IL-2 at aconcentration of 20 U/ml. This culture was maintained until day 25, withpartial renewal (50%) of the medium twice a week. The number of viablecells was quantified by a tetrazolium-based colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)method (Pauwels, R. et al J. Virol. Methods, 20,309-321, (1988)), andactivity is expressed as a percentage of the signal in the drug-free andvirus-free control. Viral replication was quantified by measuring theHIV-1 p24 antigen by ELISA using the Dupont de Nemours kit.

EXAMPLE 3

A further study demonstrated inhibition of viral spread in preactivatedPBMC culture infected with the HIV virus, by the mixture of HC and ddI.

PBMC preactivated with PHA and IL-2, were infected and treated by HC at0.15 mM; this concentration corresponds to the IC₅₀ (inhibitoryconcentration 50%) after 3 days measured by MTT tests, with cellularviability >90% (this cell viability was determined by treating the cellswith 2% Trypan Blue for 2 min and monitoring for dye exclusion). Thecombination of 0.15 mM of HC and 10 μM of ddI does not modify the IC₅₀and cellular viability.

As can be seen (FIG. 3), the virus replicated rapidly in the non-treatedculture maintaining a stable level as from day 6 (day 6=71815; day12=72750; day 20=62750 pg p24/ml). Treatment with ddI alone at 10 μM andHC alone at 0.15 mM induces inhibition of 97.1% (2071 pg p24/ml) and82.6% (12500 pg p24/ml) respectively at day 6. By contrast, a majorsynergistic effect is observed with the combination of 10 μM ddI and0.15 mM HC, with an inhibition of 99.8% (100 pg p24/ml) at day 6 and noresidual viral production detectable (<1 pg. p24/ml) at day 12 and day20.

This major synergistic effect, having been demonstrated withnon-activated lymphocytes, where the combination of ddI with HCeradicates the HIV infection from the cells, is also observed here wherelymphocytes are preactivated and treated with the combination of ddI andHC while the PBMC are replicating.

In FIG. 3, there is shown the elimination of viral replication by thecombination of HC and ddI preactivated PBMC culture infected with theHIV virus. The PBMC were purified from peripheral blood by discontinuousFicoll density gradient centrifugation. The cells were grown at adensity of 1.3×10⁶ cells/ml in RPMI 1640 medium supplemented with 10%FCS, 2 mM glutamine, penicillin 100 IU/ml and streptomycin 100 μg/ml, inthe presence of PHA at a concentration of 1 μg/ml and recombinant IL-2at 20 U/ml for 72 hours, then infected by HIV-1 strain IIIB at amultiplicity of 5000 TCID for 10⁶ cells (241 pg/ml p24 antigenequivalent of virus). In FIG. 3, the first time point represents viralinfection of the cells. After 2 hours of virus-cell contact, the cellswere washed twice and placed in the culture medium containing IL-2 butwithout PHA in the presence of ddI at a concentration of 10 μM and of HCat a concentration of 0.15 mM. These cultures were maintained for 20days, with partial renewal of the medium and of the two drugs twice aweek maintaining the initial concentration. At day 6 and day 14 freshuninfected donor PBMC were added (5×10⁵ /ml) to replenish aged cultures(Nature, 361:1993, 650-654). The number of viable cells were quantifiedby a tetrazolium-based colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)method (Pauwels, R. et al J. Virol. Methods, 20:309-321, (1988)), andactivity is expressed as a percentage of the signal in the drug-free andvirus-free control. Viral replication was quantified by measuring theHIV-1 p24 antigen by ELISA using the Dupont de Nemours kit.

Gao et al in Proc. Nat'l. Acad, Sci. U.S.A., cited supra, teach that"human immunodeficiency virus type 1 (HIV-1) viral DNA synthesis inquiescent and activated peripheral blood lymphocytes (PBLs) was studied.Incomplete viral DNA (previously demonstrated to be associated withHIV-1 virions) is carried by HIV-1 virions into quiescent and activatedPBLs, contributing to the formation of an early viral DNA pool in thesecells. The viral DNA is subsequently completed but only extremely slowlyand inefficiently in quiescent PBLs compared to that in stimulated PBLs.We find that this correlates with significantly lower levels of dNTPsubstrates in quiescent compared to activated PBLs. At these low dNTPconcentrations, HIV-1 reverse transcriptase acts in a partiallydistributive manner. Increasing dNTP concentrations from the levels ofquiescent PBLs to the levels of activated PBLs increases the processiveaction of the reverse transcriptase, which in turn stimulates rapid andefficient formation of full length DNA. Furthermore, hydroxyureatreatment of stimulated PBLs decreases the dNTP levels and the DNAsynthesis rate to levels comparable to quiescent PBLs. Our datatherefore indicate that low levels of dNTP may explain why HIV-1 DNA issynthesized slowly and inefficiently in quiescent PBLs and suggest thatpharmacologic induction of low dNTP levels represents a therapeuticapproach for inhibition of HIV-1 replication."

The explanation by Gao et al given for slow and inefficient HIV-1 viralDNA synthesis in quiescent PBLs may well be valid, given that viral DNAis synthesized completely in PBLs under conditions of sufficientreplication/activation (Fauci, 1993, Science, 262:1011-1018.).

However, Gao et al fail to explain how HC (hydroxycarbamide orhydroxyurea! by means of its inhibitory action on ribonucleotidereductase, and the reduction of dNTP pools in activated lymphocytes,would constitute a potential use of HC in the treatment of patients withAIDS. At the concentration of 1 mM, HC partially reduces the variousdNTP pools studied, see FIG. 4(a).

A comparison of table 1 (dNTP pools in quiescent and PHA stimulatedPBLs) of Gao et al, and FIG. 4(a) (effect of HC on dNTP pools inPHA-stimulated PBLs) shows that the reduction of pools levels in table 1is substantially greater than the HC-induced reduction over anequivalent 48 hour period. (See table 1).

In one accepts what Gao et al hypothesize that viral DNA synthesis in"resting" cells while show and inefficient eventually produces completeviral DNA capable of integration, it is difficult to understand how,under the conditions described, HC could have activity in AIDS patients.It is demonstrated in the present disclosure (FIG. 1) that quiescentcells in the presence of HC at nontoxic concentrations are incapable ofpreventing the production of infectious virions as measured by the HIV-1p24 antigen in the supernatant after stimulation by PHA and IL-2.

An explanation of the HC-induced depletion of the dNTP pools which"significantly reduces the rate of HIV-1 DNA synthesis and inhibits thecompletion of viral DNA synthesis in PHA stimulated PBLs", see FIG. 4(b)of Gao et al, would by that HC at the concentration of 1 mM is cytotoxicfor non-activated lymphocytes pretreated for 24 hours and activated byPHA and IL-2 for the following 48 hours in the presence of HC. Undersuch conditions, greater than 70% of the PBLs die due to the drug'stoxicity.

Under the heading "Potential use of hydroxyurea in AIDS therapy", Gao etal state that "by depleting the cellular dNTP pool, hydroxyurea inexpected to increase the therapeutic effect of nucleoside analogs3'-azido-3'-deoxythymidine, dideoxyinosine, or dideoxycytosine, whichact as competitors of cellular dNTP". If this were true, one wouldexpect that, in the treatment of infected "resting" cells where dNTPpools are found at their lowest levels, nucleoside analogs would have amajor effect. However, as shown in the present disclosure, (FIGS. 1 and2) the treatment of "resting" cells infecting by HIV-1 for seven daysand treated by AZT alone at 5 μM has only a slight effect on viralreplication as measured by p24 antigen. Similarly, for ddI alone, at thesame concentration, and under the same conditions, viral replication isonly temporarily and partially inhibited, regaining the level of theinfected control at day 25 (FIG. 1). It is not, therefore, in acceptingthe Gao et al explanation that one could have predicted an increase inthe therapeutic effect in AIDS therapy by associating a nucleosideanalog with HC, all the more so since the results in the presentdisclosure show that a surprising synergistic effect is observed for theassociation of HC and ddI, but not at all for HC and AZT.

The subject of the present invention is also a new composition for thetreatment of a cell population in the presence of a retrovirus.Additionally, the invention includes a pharmaceutical compositionintended, in particular, for the treatment and prevention of retroviralinfections, especially those linked to HIV and AIDS wherein thecomposition contains a synergistic combination of hydroxycarbamide (HC)and a reverse transcriptase inhibitor, in particular a synergisticcombination of a dideoxynucleoside except AZT and hydroxycarbamide, mostpreferably a synergistic combination of dideoxyinosine andhydroxycarbamide as active principle, in a pharmaceutically acceptablevehicle. The composition of the invention can also contain inert orpharmacodynamically active additives, carriers and/or excipients.

The pharmaceutical composition of the invention can take the form of alypholized powder of the active substance, to be dissolved immediatelybefore use in a physiological solution for the purpose of injection. Themedicament can then be administered parenterally, for exampleintravenously, intraperitoneally, in the cerebrospinal fluid, and thelike. For injection, the active principle is dissolved in aphysiological solution until the desired concentration foradministration is obtained.

The pharmaceutical composition according to the invention can also takea form which is suitable for oral administration. For example, suitableforms are tablets, hard gelatin capsules, dragees, powders and granules.The formation of such oral forms is well-known to those skilled in theart. Any of the known formulations are useful in preparing the instantoral pharmaceutical compositions.

As regards the dosage of the medicament according to the invention, itwill be clear to the artisan that the doses to be administered arevariable according to the treatment period, and frequency ofadministration, the host, and the nature and severity of the disease andthat the dosages can be easily determined without any undue amount ofexperimentation.

The compositions of the invention are administered in substantiallynon-toxic dosage concentrations sufficient to insure the release of asufficient dosage unit of the present synergistic combination into thepatient to provide the desired inhibition of the spread of theretrovirus. The actual dosage administered will be determined byphysical and physiological factors such as age, body weight, severity ofcondition, and/or clinical history of the patient. With theseconsiderations in mind, the dosage of the instant synergisticcombination for a particular subject can be readily determined by thephysician. It might be noted that in extreme cases a dosage approachingthe toxic level may be the acceptable treatment protocol.

For example, in the treatment of HIV-infected and AIDS patients, thecomposition can comprise from about 1 to 66 mg/Kg/day of ddI and fromabout greater than 5 mg/Kg/day to about 20 mg/Kg/day of HC.

The invention also covers the use of hydroxycarbamide (HC) anddideoxyinosine in combination with other medical compositions intendedfor the treatment of retroviral infections and tumors. Immunostimulantsand immunomodulators such as for example cytokines, including IL-2,IL-12 and interferon molecules can be used in combination with thepresent invention.

A preferred range for in vitro administration of the compositions of thepresent invention includes hydroxycarbamide in a concentration greaterthan 0.05 mM and less than or equal to 0.25 mM in combination with adideoxynucleoside except AZT such as ddI at concentrations which aregenerally known and used in the art. A preferred embodiment of thepresent invention utilizes HC at 0.15 mM and the dideoxynucleoside suchas ddI in a range of between about 0.01 μM to about 100 μM, preferablybetween about 2.5 μM to about 25 μM, most preferably from about 5 μM toabout 10 μM.

All of the references cited hereinabove are expressly incorporatedherein, in toto, by reference thereto.

The invention has been described with reference to specific andpreferred embodiments. It will be recognized by those skilled in the artthat numerous changes and substitutions may be made without departingfrom the spirit and scope of the invention.

We claim:
 1. A method of treating an HIV infected human comprisingadministering to said human a dosage of hydroxycarbamide (HC) to yieldan in vivo blood plasma concentration of about 0.15 mM and a dosage of2', 3'-dideoxyinosine (ddI) to produce an in vivo blood plasmaconcentration in the range of about 5-10 μM.
 2. A method of treating anHIV infected human comprising administering to said human a dosage ofhydroxycarbamide (HC) to yield an in vivo blood plasma concentration ofabout 0.15 mM and a dosage of 2', 3'-dideoxyinosine (ddI) to produce anin vivo blood plasma concentration of about 5 μM.
 3. A method oftreating an HIV infected human comprising administering to said human adosage of hydroxycarbamide (HC) to yield an in vivo blood plasmaconcentration of about 0.15 mM and a dosage of 2', 3'-dideoxyinosine(ddI) to produce an in vivo blood plasma concentration of about 10 μM.